We have established assays to examine expression of the drug resistance phenotype using flow cytometric analysis of intact single cells. There are significant advantages to this approach, including the ability to derive rapid, semiquantitative data on tens of thousands of cells and the ability to study the heterogeneity of drug resistance within clinical specimens. The techniques developed include analysis of P-glycoprotein expression using the monoclonal antibody MRK-16 analysis of the expression of thymidylate synthetase using a monoclonal antibody developed by Dr. Patrick Johnson, and analysis of the expression of the folate binding protein and properties of the reduced folate transporter, using fluoresceinated methotrexate. In the past year, we have used these techniques in a wide variety of applications in collaborative studies. These applications include examination of the reduced folate transporter in breast cancer cell lines; expression of P-glycoprotein in breast cancer cell lines transfected with the mdr 1 gene and the gene for protein kinase C alpha; comparison of flow cytometry with Western blot, Northern blot, and RNA in situ for the analysis of P-glycoprotein in colon cancer cell lines expressing clinically relevant levels of P-glycoprotein and drug resistance; examination of the expression of the folate binding protein in CHO, MCF-7, and L cells transiently transfected with a folate binding protein expression vector; analysis of thymidylate synthetase expression in colon cancer cell lines; and analysis of the cell cycle phase distribution after treatment of human colon cancer cells with the novel antimetabolite MRPP.